Ribotoxins are potent inhibitors of protein synthesis. They are believed to act as enzymes on the 28S rRNA of the eucaryotic ribosome and can be divided into two classes (Jiminez, A et al., 1985, Ann. Rev Microbiol., 39, 649-672, and Wool, I. G. et al., 1990, in Hill, W. E. et al. (ed.) The Ribosome Structure, Function & Evolution, American Society of Microbiology, Washington DC, 203-214). Restrictocin, mitogillin and .alpha.-sarcin belong to the class which cleaves a single phosphodiester bond. Ricin belongs to the other class which cleaves an N-glycosidic linkage between base and ribose. The nucleotide sequence of the restrictocin gene has been described by Lamy, B. & Davies, J. in Nucleic Acids Research, 1991, 19, 1001-1006.
Such toxins may be used for example in the preparation of immunotoxins for cancer therapy. Pharmaceutical applications of toxins require access to relatively large quantities of toxin of a consistent quality. Cost-effective production techniques are of considerable commercial importance. Toxins may be obtained from their natural sources but this is undesirable because of poor yields and batch to batch variability leading to inconsistent product quality and high production cost. Natural sources are unsuitable for production of analogues of toxins which may be required for certain applications.
Genetic engineering techniques may be applied to the production of toxins but in the case of restrictocin, mitogillin and .alpha.-sarcin known methods have failed to provide a cost-effective or high yielding production method. Restrictocin expression using recombinant DNA technology has been described by Lamy, B. and Davies, J. (Nucleic Acids Res. (1991) 19(5) p1001-1006. They expressed and secreted restrictocin from Aspergillus nidulans, but the level of secretion was low, even lower than that from the natural producer strain, Aspergillus restrictus. There have been no reports of restrictocin expression in bacteria, although the related toxin, alpha sarcin, has been expressed in E. coli (Henze, P-P, C. et al. (1990) Eur. J. Biochem. 192, p127-131). Again, expression was low, as the protein could not be detected in total cell lysates by Coomassie blue staining of one-dimensional SDS PAGE electrophoretograms.
In both the Henze and Lamy documents expression vectors designed to allow secretion were employed. This allowed the authors to circumvent toxic effects on the host cells. Henze teaches that a secretion system is necessary, even in E. coli, to avoid toxic effects on the host cell (see 2nd. paragraph of the Discussion on page 130) and also teaches that the host ribosomes are sensitive in vivo to the expressed toxin (see 7th paragraph of the Discussion on page 130). Lamy teaches that even one free molecule of restrictocin inside an Aspergillus cell may be lethal (see 7th line of the first column on page 1005). Hence known methods teach that if expression vectors designed to give high level intracellular accumulation of restrictocin were used the method would not work due to toxicity of the product leading to cell death.